Rini Rossanti1,2, Tomoko Horinouchi1, Tomohiko Yamamura1, China Nagano1, Nana Sakakibara1, Shinya Ishiko1, Yuya Aoto1, Atsushi Kondo1, Sadayuki Nagai1, Eri Okada1, Shingo Ishimori1, Hiroaki Nagase1, Satoshi Matsui3, Keiichi Tamagaki4, Yoshifumi Ubara5, Masahiko Nagahama6, Yuko Shima7, Koichi Nakanishi8, Takeshi Ninchoji1, Masafumi Matsuo9, Kazumoto Iijima10,11, Kandai Nozu1. 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan. 2. Department of Child Health, Nephrology Division, Dr. Hasan Sadikin General Hospital/Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia. 3. Department of Nephrology and Hypertension, Mitsubishi Kyoto Hospital, Kyoto, Japan. 4. Department of Nephrology, Kyoto Prefectural University of Medicine, Kyoto, Japan. 5. Nephrology Center, Okinaka Memorial Institute for Medical Research, Tokyo, Japan. 6. Internal Medicine, St. Luke's International Hospital, Tokyo, Japan. 7. Department of Pediatrics, Wakayama Medical University, Wakayama, Japan. 8. Department of Child Health and Welfare (Pediatrics), Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan. 9. Research Center for Locomotion Biology, Kobe Gakuin University, Kobe, Japan. 10. Hyogo Prefectural Kobe Children's Hospital, Kobe, Japan. 11. Department of Advanced Pediatric Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.
Abstract
Background: Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. Methods: We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome (COL4A3 [c.693G>A (p.Val231=)] and COL4A4 [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]). Results: Both in vivo and in vitro splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing. Conclusions: This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.
Background: Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. Methods: We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome (COL4A3 [c.693G>A (p.Val231=)] and COL4A4 [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]). Results: Both in vivo and in vitro splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing. Conclusions: This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.
Authors: Vladimir Presnyak; Najwa Alhusaini; Ying-Hsin Chen; Sophie Martin; Nathan Morris; Nicholas Kline; Sara Olson; David Weinberg; Kristian E Baker; Brenton R Graveley; Jeff Coller Journal: Cell Date: 2015-03-12 Impact factor: 41.582
Authors: Sue Richards; Nazneen Aziz; Sherri Bale; David Bick; Soma Das; Julie Gastier-Foster; Wayne W Grody; Madhuri Hegde; Elaine Lyon; Elaine Spector; Karl Voelkerding; Heidi L Rehm Journal: Genet Med Date: 2015-03-05 Impact factor: 8.822